蝴蝶蘭基因轉殖I -暫時性基因表現之最佳條件

Phalaenopsis Orchid Gene Transformation (I) - Optimization of Transient Gene Expression

尋找基因轉殖的最佳條件是獲得轉基因植株的第一步。但是，到目前為止，還沒有任何轉殖系統可以成功地將外來基因轉入蝴蝶蘭的類圓球體。利用蝴蝶蘭的類圓球體作為材料，我們分別檢測基因槍轉殖法與農桿菌轉殖系統的效果，並尋找能獲得最佳暫時性表現的條件。對於基因槍轉殖法，我們嘗試了各種條件，包括擊發次數、墊片（rupture disk）、培植體與macrocarrier之間的距離以及擊發時是否有濾紙介於培植體與培養基之間等。最適合用於類圓球體的條件則是將培植體直接置於培養基上，利用650psi的壓力及距離macrocarrier 9公分擊發一次。至於農桿菌轉殖系統，我們則測試了在農桿菌共培養的過程中加入了不同時間的超音波震盪以及各種造成類圓球體受傷的方法。最適合的條件是將類圓球體浸泡於農桿菌液中，並進行超音波震盪150秒。本研究的結果提供了蘭花轉殖系統的建立與獲得基因轉殖蘭花的基礎。

The ensured optimal conditions for gene transfer is a prerequisite for successful development of transgenic plants. To date, no suitable method has been established for genetic transformation of Phalaenopsis orchid protocorm-like body (PLB). Using this material, we optimized parameters by two major genetic transformation methods: Agrobacterium-mediated and particle bombardment gene. Optimal penetration of particles into the PLB was in the biolistic apparatus was achieved by evaluating times of bombardment, the strength of rupture disk, distance of the rupture disk between the macro-carrier and the samples. During shooting, the PLB was placed on a filter paper. The best GUS expression was observed when PLBs were cultured without the filter paper base and bombarded once at 650 psi, 9cm. In the bio-genetic transfer system, we evaluated the effects of the duration of sonication and various wounding treatments during Agrobacterium infection on orchid PLBs. Maximum GUS expression was obtained when Taisuco 339 (TS339) PLBs were sonicated for 150 sec. in the presence of Agrobacterium followed by co-cultivation on T2 medium for 3 days at 26o C. These results provide the basis for the optimal transgenic development of transgenic orchid plants.